Homologous recombination is a critically important process responsible for repairing double-strand DNA breaks and exchanging genetic material between homologous chromosomes in cells undergoing meiosis. Our goal is to understand the mechanisms of homologous recombination in humans. The mechanism of homologous recombination involves forming a cross-stranded structure between two homologous chromosomes called a Holliday junction. The Holliday junction possesses a remarkable property, in which this structure can freely translocate along the DNA axis in a process termed branch migration. The central role of branch migration in homologous recombination and DNA repair has only recently been fully appreciated. Branch migration drives 1) the resolution of homologous recombination intermediates, 2) the retrieval of information lost at sites of DNA damage, and 3) the restart of stalled DNA replication forks. In human cells, branch migration is catalyzed by RecQ helicases, including BLM and WRN. Mutations in the BLM and WRN genes cause a high degree of genome instability, which lead to premature aging disorders called Bloom's and Werner's Syndromes. Recent genetic analysis has demonstrated that Rad54 also plays an important role in homologous recombination of eukaryotes. Double RAD54 BLM knockouts show a dramatic increase in chromosomal instability over the level observed in either single knockout, indicating a functional redundancy between these two proteins. Indeed, our lab recently discovered that Rad54 can also promote branch migration of Holliday junctions. We will use purified human proteins in an array of biochemical assays specifically designed to investigate the branch migration activity of Rad54 and BLM. The goals of this proposal are to understand how Rad51 affects the function of Rad54 and BLM in homologous recombination and investigate the non-canonical helicase activity of Rad54 that functions during branch migration. These assays will be combined with a mutational analysis of Rad54 to determine which motifs are crucial to its biological activities. Our goal is to better understand how the branch migration activities of these two similar, but distinct enzymes contribute to the maintenance of genome integrity in human cells.